Antigenic relationship between Pseudomonas Bal-31 membranes and bacteriophage PM2

Summary Antisera against bacteriophage PM2 and against membranes of its host cell, Pseudomonas BAL-31, were prepared. Cross-reactivity between these two antigens and both antisera was found by immunodiffusion, complement fixation and viral neutralization experiments. Anti-membrane sera up to a dilution of 1/100 were able to neutralize 60% of the infective capacity of PM2. This neutralizing capacity was partially abolished by the presence of Pseudomonas BAL-31 membranes. It is concluded that similar antigenic determinants are present in the PM2 phage and in the host membrane.     

Origin of Phospholipid in Bacteriophage PM2

Phosphatidylethanolamine is the only phosphatide present in purified bacteriophage PM2 when obtained from bacteria grown and infected in minimal medium. The growth cycle of bacteriophage PM2 shows the basic general features described for virulent bacteriophages. PM2 infection of Pseudomonas BAL-31 causes a pronounced increase in deoxyribonucleic acid synthesis, but no detectable effect on the incorporation of phosphorus into lipid. When (32)P-prelabeled bacteria are infected with PM2, the specific activity of the phosphatide phosphorus in the virus is almost the same as that in the host bacteria labeled before infection. It is concluded that the viral phosphatide is not synthesized de novo after infection, but it probably is derived from preexisting cellular phosphatidylethanolamine.     

Effect of lipid alkyl chain perturbations on the assembly of bacteriophage PM2.

The lipid-containing bacteriophage PM2 can produce infectious virus in cultures infected at temperatures up to 31.5 degrees C, but not at 34 degrees C. Its host, Pseudomonas BAL-31, grows at 34 degrees C and cultures infected at that temperature undergo lysis. Sucrose-gradient analysis shows that 34 degrees C lysates contain no PM2-like particles. Temperature-shift experiments establish that the thermally sensitive process is late in infection when virus assembly is taking place. Adamantanone, a small hydrophobic molecule that perturbs membrane hydrocarbon zones, prevents the production of infective virus. Concentrations which prevent virus production have no effect on host-cell growth or stability of mature virions. Adamantanone exerts its effects late in the infectious cycle, and lysates amde in its presence contain no PM2-like particles. These experiments, carried out at 25 degrees C, indicate that adamantanone prevents the assembly of stable PM2 virus. Spin-label studies suggest that the lipid alkyl chains of the host-cell membrane are in an "ordered" state at temperatures below about 33 degrees C and undergo a transition to a "disordered" state above that temperature. Furthermore, the addition of adamantanone perturbs the hydrocarbon zones, producing a greater degree of disorder even below 25 degrees C. Our findings suggest that the cell membrane can function and grow with the lipid alkyl chains in either the "ordered" or "disordered" state, but that the "ordered" state must be maintanined for PM2 assembly to occur.     

Effect of lipid alkyl chain perturbations on the assembly of bacteriophage PM2

The lipid-containing bacteriophage PM2 can produce infectious virus in cultures infected at temperatures up to 31.5 °C, but not at 34 °C. Its host, Pseudomonas BAL-31, grows at 34 °C and cultures infected at that temperature undergo lysis. Sucrose-gradient analysis shows that 34 °C lysates contain no PM2-like particles. Temperature-shift experiments establish that the thermally sensitive process is late in infection when virus assembly is taking place.Adamantanone, a small hydrophobic molecule that perturbs membrane hydrocarbon zones prevents the production of infective virus. Concentrations which prevent virus production have no effect on host-cell growth or stability of mature virions. Adamantanone exerts its effects late in the infectious cycle, and lysates made in its presence contain no PM2-like particles. These experiments, carried out at 25 °C, indicate that adamantanone prevents the assembly of stable PM2 virus.Spin-label studies suggest that the lipid alkyl chains of the host-cell membrane are in an “ordered” state at temperatures below about 33 °C and undergo a transition to a “disordered” state above that temperature. Furthermore, the addition of adamantanone perturbs the hydrocarbon zones, producing a greater degree of disorder even below 25 °C. Our findings suggest that the cell membrane can function and grow with the lipid alkyl chains in either the “ordered” or “disordered” state, but that the “ordered” state must be maintained for PM2 assembly to occur.     

Calcium requirement for assembly of the lipid-containing bacteriophage PM2

The bacteriophage PM2 requires extracellular Ca²⁺ at concentrations greater than 3 · 10⁻⁴ M for the production of viable virus, whereas the host cell Pseudomonas BAL-31 grows normally in medium containing 3 · 10⁻⁵ M Ca²⁺ (low calcium). Virus attachment occurs normally in low calcium, the infected cultures partially lyse, but no infectious virus particles are released. Sucrose gradient analysis shows that lysates made in low calcium contain no PM2-like particles. The addition of calcium very late in the infectious cycle completely restores virus production to cultures infected in low calcium, whereas removal of calcium after infection prevents virus production. Our experiments indicate that Ca²⁺ is essential for some process late in the lytic cycle, such as the final assembly of stable, infectious PM2 particles.     

Transbilayer distribution of phospholipids in bacteriophage membranes

We have previously demonstrated that the membranes of several bacteriophages contain more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host membrane from where they are derived. Here, we determined the transbilayer distribution of PG and PE in the membranes of bacteriophages PM2, PRD1, Bam35 and phi6 using selective modification of PG and PE in the outer membrane leaflet with sodium periodate or trinitrobenzene sulfonic acid, respectively. In phi6, the transbilayer distributions of PG, PE and cardiolipin could also be analyzed by selective hydrolysis of the lipids in the outer leaflet by phospholipase A(2). We used electrospray ionization mass-spectrometry to determine the transbilayer distribution of phospholipid classes and individual molecular species. In each bacteriophage, PG was enriched in the outer membrane leaflet and PE in the inner one (except for Bam35). Only modest differences in the transbilayer distribution between different molecular species were observed. The effective shape and charge of the phospholipid molecules and lipid-protein interactions are likely to be most important factors driving the asymmetric distribution of phospholipids in the phage membranes. The results of this first systematic study on the phospholipid distribution in bacteriophage membranes will be very helpful when interpreting the accumulating high-resolution data on these organisms.     

Transbilayer distribution of phospholipids in bacteriophage membranes

We have previously demonstrated that the membranes of several bacteriophages contain more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host membrane from where they are derived. Here, we determined the transbilayer distribution of PG and PE in the membranes of bacteriophages PM2, PRD1, Bam35 and phi6 using selective modification of PG and PE in the outer membrane leaflet with sodium periodate or trinitrobenzene sulfonic acid, respectively. In phi6, the transbilayer distributions of PG, PE and cardiolipin could also be analyzed by selective hydrolysis of the lipids in the outer leaflet by phospholipase A₂. We used electrospray ionization mass-spectrometry to determine the transbilayer distribution of phospholipid classes and individual molecular species. In each bacteriophage, PG was enriched in the outer membrane leaflet and PE in the inner one (except for Bam35). Only modest differences in the transbilayer distribution between different molecular species were observed. The effective shape and charge of the phospholipid molecules and lipid-protein interactions are likely to be most important factors driving the asymmetric distribution of phospholipids in the phage membranes. The results of this first systematic study on the phospholipid distribution in bacteriophage membranes will be very helpful when interpreting the accumulating high-resolution data on these organisms.     

Effects of temperature and host cell genetic characteristics on the replication of the lipid-containing bacteriophage PR4 in Escherichia coli.

The lipid-containing bacteriophage PR4 is of special intest because it can replicate in various gram-negative bacteria, including Escherichia coli, that carry one of a group of drug resistance plasmids. PR4 grown in E. coli strain PS2R contains about 10% lipid by weight, with the negatively charged phospholipid phosphatidylglycerol being the most abundant lipid in the virion. We now report the following. (i) PR4 attaches to E. coli with an attachment rate constant of Ka approximately 6.2 X 10(-10) ml/min, which is about twice that of the enveloped phage phi6 (to Pseudomonas phaseolicola), but a factor of 5 less than that of phage PM2 (to Pseudomonas BAL-31). (ii) Use of an E. coli glycerol auxotroph indicated that a normal amount of PR4 replication occurs only if glycerol starvation (inhibition of all phospholipid synthesis) begins no earlier than about halfway through the lytic cycle. (iii) Use of an E. coli fatty acid synthesis temperature-sensitive mutant and an E. coli phosphatidylethanolamine synthesis temperature-sensitive mutant indicate that PR4 replication can occur in the absence of either normal fatty acid synthesis or normal phospholipid synthesis if the infection takes place prior to the termination of overall cell growth and the onset of cell death, (iv) Whereas PR4 burst size in nutrient media at 30 degrees C to 42%C is about 40, the burst size at 20 degrees C is less than 3, Temperature-shift experiments show that the temperature late in infection determines the burst size.     

Infection of spheroplasts of Pseudomonas with DNA of bacteriophage PM2

Summary Spheroplasts of Pseudomonas BAL-31/PM2, obtained by treatment of the bacteria with lysozyme, can be infected with purified DNA from bacteriophage PM2. After 4 h of incubation the yield of progeny phage reaches a value of 10⁷-6×10⁷ plaque forming units/g PM2 DNA. The yield increases linearly with the concentration of DNA over at least 3 orders of magnitude.The biological activity of double-stranded circular PM2 DNA containing one or more single-strand breaks per molecule (component II), does not differ significantly from that of intact PM2 DNA (component I). Single-stranded PM2 DNA obtained by denaturation of component II, and the irreversible alkali-denatured form of component I are also infective.     

Role of the pneumococcal autolysin (murein hydrolase) in the release of progeny bacteriophage and in the bacteriophage-induced lysis of the host cells.

The pneumococcal bacteriophage Dp-1 seems to require the activity of the N-acetylmuramic acid-L-alanine amidase of the host bacterium for the liberation of phage progeny into the medium. This conclusion is based on a series of observations indicating that the exit of progeny phage particles is prevented by conditions that specifically inhibit the activity of the pneumococcal autolysin. These inhibitory conditions are as follows: (i) growth of the bacteria on ethanolamine-containing medium; (ii) growth of the cells at pH values that inhibit penicillin-induced lysis of pneumococcal cultures and lysis in the stationary phase of growth; (iii) addition of trypsin or the autolysin-inhibitory pneumococcal Forssman antigen (lipoteichoric acid) to the growth medium before lysis; (iv) infection of an autolysin-defective pneumococcal mutant at a multiplicity of infection less than 10 (treatment of such infected mutant bacteria with wild-type autolysin from without can liberate the entrapped progeny phage particles); (v) release of phage particles and culture lysis can also be inhibited by the addition of chloramphenicol to infected cultures just before the time at which lysis would normally occur. Bacteria infected with Dp-1 under conditions nonpermissive for culture lysis and phage release secrete into the growth medium a substantial portion of their cellular Forssman antigen in the form of a macromolecular complex that has autolysin-inhibitory activity. We suggest that a phage product may trigger the bacterial autolysin by a mechanism similar to that operating during treatment of pneumococci with penicillin (Tomasz and Waks, 1975).     

An alkyl imidate labeling study of the organization of phospholipids and proteins in the lipid-containing bacteriophage PR4

The structure of the lipid-containing bacteriophage PR4 was studied using two alkyl imidates, ethyl acetimidate (EAI), a reagent permeant to lipid bilayers and isethionyl acetimidate (IAI), which is impermeant to membranes. The virion is an icosahedral particle consisting of a protein coat surrounding a membrane of phospholipid and protein which in turn encloses the DNA genome. Upon exposure to the permeant reagent, EAI, 50% of the phosphatidylethanolamine (PE) molecules reacted rapidly (half-life less than 10 min). A similar fraction of the PE also reacted with IAI, the impermeant reagent. The remaining half of the PE molecules reacted slowly with EAI (half-life of 80 min) and failed to react with IAI. All of the phage proteins reacted with both EAI and IAI (except a DNA-associated protein which reacted only with EAI). These labeling results indicate that the phage membrane consists of a lipid bilayer and that at least a portion of each phage protein (except the DNA-associated protein) is exposed on the external face of the lipid bilayer. Several of the membrane proteins could be cross-linked either to the phage membrane PE after EAI treatment or to phage phosphatidylglycerol after periodate treatment. The major structural protein of the phage was readily cross-linked to PG but failed to cross-link to PE suggesting that the protein specifically interacts with PG.     

Loss of adsorptive ability--the reason for inactivation of the RM2 bacteriophage during storage

The kinetics of bacteriophage PM2 inactivation at storage was compared with the kinetics of bacteriophage adsorption on Alteromonas espejiana BAL-31 host cells. Adsorption ability and infectivity are lost with the same rate at temperatures 4-28 degrees C suggesting the loss of adsorption ability to result in bacteriophage inactivation. At higher temperatures infectivity is lost more rapidly than the ability of adsorption. The single hit kinetics of adsorption ability loss suggests the simple model of independent inactivation of 12 antireceptors located at the tops of icosaedric capsid to be erroneous. At bacteriophage inactivation the major port of protein I, a fragment of antireceptors, is preserved in the capsid composition.     

Induction and general properties of beta-galactosidase and beta-galactoside permease in Pseudomonas BAL-31.

The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside (ONPG) by BAL-31, a marine Pseudomonas that acts as a host for bacteriophage PM2, was studied with intact cells and with cell-free extracts. A transport system for ONPG in whole cells and a beta-galactosidase activity in extracts were evident for cells grown on lactose minimal medium. It was found that the addition of isopropylthio-beta-D-galactopyranoside (IPTG) to cells growing in rich medium induced an ONPG hydrolytic activity detectable in cell extracts but cryptic in whole cells. The existence of a transport system for IPTG, which remained cryptic for ONPG, became apparent from studies of the rates of induction of beta-galactosidase as a function of cell mass at different concentrations of IPTG. The main properties of beta-galactosidase and the lactose transport system of BAL-31 were studied in terms of how they were affected by pH, temperature, or by the presence of several sugars. IPTG competitively inhibits the hydrolysis of ONPG by cell extracts. In cells pregrown on lactose, IPTG slightly inhibits the transport of ONPG. Glucose, and with less efficiency lactose, also inhibits the hydrolysis of ONPG in cell extracts. The growth of cells on lactose minimal medium was inhibited by the addition of IPTG. A mechanism for this inhibition and for the inhibition of ONPG transport by IPTG is discussed.     

A novel lysis system in PM2, a lipid-containing marine double-stranded DNA bacteriophage

In this study we investigated the lysis system of the lipid-containing double-stranded DNA bacteriophage PM2 infecting Gram-negative marine Pseudoalteromonas species. We analysed wt and lysis-deficient phage-induced changes in the host physiology and ascribed functions to two PM2 gene products (gp) involved in lysis. We show that bacteriophage PM2 uses a novel system to disrupt the infected cell. The novelty is based on the following findings: (i) gp k is needed for the permeabilization of the cytoplasmic membrane and appears to play the role of a typical holin. However, its unique primary structure [53 aa, 1 transmembrane domain (TMD)] places it into a new class of holins. (ii) We have proposed that, unlike other bacteriophages studied, PM2 relies on lytic factors of the cellular origin for digestion of the peptidoglycan. (iii) gp l (51 aa, no TMDs) is needed for disruption of the outer membrane, which is highly rigidified by the divalent cations abundant in the marine environment. The gp l has no precedent in other phage lytic systems studied so far. However, the presence of open reading frame l-like genes in genomes of other bacterial viruses suggests that the same system might be used by other phages and is not unique to PM2.     

Effect of Shift-Down and Growth Inhibition on Phospholipid Metabolism of Escherichia coli

The metabolism of phospholipids of Escherichia coli was studied under conditions which inhibit various metabolic processes. Phospholipid synthesis and turnover were not inhibited by growth-inhibitory amounts of various antibiotics. Turnover of phosphatidylglycerol (PG) was inhibited by small amounts of dinitrophenol and by anaerobiosis. Turnover of phosphatidylethanolamine (PE), which is not detected in control cultures, was demonstrated under conditions of incipient lysis. When cells were shifted down from a rich to a poor medium, PE synthesis was inhibited, and incorporation of glycerol into the distal position of PG was stimulated. Under these conditions, turnover of the phosphate and the acylated glycerol moieties of PG was inhibited. Increased synthesis of PE was detected when filamentous cells were induced to make septa. The results indicate that PE synthesis is related to growth and cell division, whereas PG metabolism is related to other cell processes.     

A novel method of transfection of marine bacteria Pseudoalteromonas espejiana with deoxyribonucleic acid of bacteriophage PM2

DNA of bacteriophage PM2 is a convenient test object for studying DNA-damaging genotoxic agents. The extent of DNA damage can be estimated by the ability of damaged DNA for transfection of host cells, marine bacterium Pseudoalteromonas espejiana (Pae), str. BAL-31. The efficiency of transfection of Pae lines maintained for long periods without freezing was found to be very low upon the use of a widely accepted transfection method developed by van der Schans et al. (1971). Such cultures grown in a medium with 10 mM Ca2+ standard for Pae contained cell aggregates and exopolymer material. Pae was found to be capable of growing in a medium without the calcium supplement in the presence of chelator EGTA (low-calcium medium, LCM). After growth in LCM, cells did not aggregate, cultures lacked the activity of nuclease BAL, and transfection efficiency of cells grown in LCM drastically increased. Based on these results, a novel procedure of transfection with an efficiency of 2 x 10(4)-2 x 10(5) infectious centers per microgram of PM2 DNA was developed.     

Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems.

Bacteriophage 80 alpha did not increase in number in cultures containing less than about 1.0 X 10(4) to 1.5 X 10(4) CFU of Staphylococcus aureus per ml, but bacteriophage replication did occur when the number of bacteria exceeded this density, either initially or as a result of host cell multiplication. The minimum density of an asporogenous strain of Bacillus subtilis required for an increase in the number of bacteriophage SP beta cI was about 3 X 10(4) CFU/ml. The threshold density of Escherichia coli for the multiplication of bacteriophage T4 was about 7 X 10(3) CFU/ml. In the presence of montmorillonite, bacteriophage T4 did not increase in number until the E. coli population exceeded 10(4) CFU/ml. The mineralization of glucose was not affected in E. coli cultures inoculated with a low number of bacteriophage T4, but it could not be detected in cultures inoculated with a large number of phage. The numbers of bacteriophage T4 and a bacteriophage that lyses Pseudomonas putida declined rapidly after being added to lake water or sewage. We suggest that bacteriophages do not affect the number or activity of bacteria in environments where the density of the host species is below the host cell threshold of about 10(4) CFU/ml.     

Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems

Bacteriophage 80 alpha did not increase in number in cultures containing less than about 1.0 X 10(4) to 1.5 X 10(4) CFU of Staphylococcus aureus per ml, but bacteriophage replication did occur when the number of bacteria exceeded this density, either initially or as a result of host cell multiplication. The minimum density of an asporogenous strain of Bacillus subtilis required for an increase in the number of bacteriophage SP beta cI was about 3 X 10(4) CFU/ml. The threshold density of Escherichia coli for the multiplication of bacteriophage T4 was about 7 X 10(3) CFU/ml. In the presence of montmorillonite, bacteriophage T4 did not increase in number until the E. coli population exceeded 10(4) CFU/ml. The mineralization of glucose was not affected in E. coli cultures inoculated with a low number of bacteriophage T4, but it could not be detected in cultures inoculated with a large number of phage. The numbers of bacteriophage T4 and a bacteriophage that lyses Pseudomonas putida declined rapidly after being added to lake water or sewage. We suggest that bacteriophages do not affect the number or activity of bacteria in environments where the density of the host species is below the host cell threshold of about 10(4) CFU/ml.     

Phospholipid Synthesis in Escherichia coli Infected with T4 Bacteriophages

After infection of Escherichia coli with T4 phage, phospholipid synthesis continued but at a reduced rate. The same phospholipid components were synthesized as in uninfected cells; however, the relative rates of (32)P(i) incorporation into phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) were altered. This alteration was most pronounced during the first 10 min after infection. Under these conditions, the isotope incorporated into PG equaled or exceeded that found in PG from uninfected cells. Chloramphenicol (CM) added before, but not 5 min after, infection inhibited the relative increase in PG synthesis, and CM added at different times after infection indicated that a protein synthesized between 3 and 6 min was required for this change to occur. Supplies of exogenous l-serine or l-alpha-glycerol-P failed to affect the relative rates of (32)P(i) incorporation into PG and PE by infected or uninfected cells. Phospholipid synthesis was somewhat higher after infection with T4rII mutants than after infection with wild-type phage. After infection with these mutants or several amber mutants, the relative synthesis of PG and PE was characteristic of T4r(+)-infected cells. The phospholipid synthesized after infection did not rapidly turn over, but infection accelerated the loss of PG synthesized prior to infection.     

Metabolic characterization of the viable, residually dividing and nondividing cell classes of recombination-deficient strains of Escherichia coli.

The abilities of rec+, recB- recC-, recA-, and recA- recB- rec C- strains to support growth of bacteriophage T4, to take up oxygen, and to maintain cell integrity have been measured. (i) With respect to bacteriophage T4 growth, T4 phage is produced with identical lysis time in single -step growth curves with all strains tested. rec- strains show reduced phage production (fewer infected centers), but the average burst size per infected center is the same for all strains tested. Some rec- cells are unable to produce any phage, whereas the remainder of the rec-cells produce phage as rapidly and as efficiently as rec+ cells. (ii) With respect to oxygen consumption, rec- strains are deficient relative to the rec+ control to the same extent as the deficiency in phage production by theculture. The reduction in oxygen consumption is coordinate with reduction in rate of mass increase of the rec- culture. (iii) With respect to cell integrity, rec- cultures show increased lysis as measured by release of beta-galactosidase into the medium. The kinetics of release suggest that rec- nondividing cells all eventually lyse. These results are most consistent with the idea that rec- residually dividing cells and viable cells are metabolically normal according to the parameters we have measured, whereas nondividing cells are metabolically inactive.